Cell barcoding for single cell sequencing

ABSTRACT

Methods and compositions for attaching cell-specific barcodes without formation of partitions is provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/930,288, entitled “CELL BARCODING FOR SINGLE CELL SEQUENCING” andfiled Nov. 4, 2019, the entire contents being incorporated herein byreference for all purposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file 094868-1212543-117410US_SL.txtcreated on Jan. 4, 2021, 2,160 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND OF THE INVENTION

Current droplet microfluidic approaches achieve thousands of cellsthroughput.

However, larger numbers can be difficult to achieve. Usingmicrofluidics, there are at least three factors that contribute to cellsuspension dead volume and, therefore, a loss of cells for analysis: 1)leftover liquid in the inlet of the microfluidic device 2) leftoverliquid in the microfluidic channels pre-partitioning and 3) material notcollected from the outlets of the microfluidic device. Moreover, verylarge droplet emulsion volumes, corresponding to higher cell throughputexperiments, are difficult to generate in a timely fashion due to thevolumes required. This may have a negative impact on cell viability andlabile nucleic acid substrates contained within cells, such as RNA.Lastly, when larger droplet emulsions are generated, their increasedvolume makes it difficult to load a single emulsion into a single tubecompatible with a thermal cycler, thus further limiting large emulsionvolume implementation.

Single-cell barcoding platforms require costly microfluidics (chips,oils, and instruments) and barcode beads. Instrumentation also requirescostly field service engineers for maintenance and to resolve hardwareissues.

Although droplet microfluidics improve scalability, only one solutionadd is readily supported unless more complicated on-chip droplet mergingand picoinjection functionalities are used.

Labeling cells directly with oligonucleotides is an option to usingoligo-conjugated beads. However, the maximum number of oligonucleotidesloaded onto a cell without disrupting cell physiology is approximately1-10 million. In droplets that are nL in size, the final concentrationof oligonucleotides will often not be sufficient to drive molecularbiology reactions hence preventing droplet compatibilities with directoligonucleotide-labeled cells.

BRIEF SUMMARY OF THE INVENTION

A high density of oligonucleotides ranging up to 10⁶ to 10⁷ moleculescan be attached to cells by a variety of methods. For example, cells canbe labeled by cell-specific oligonucleotide-conjugated antibodies(Stoeckius et al 2018, Genome Biology) or by lipid-modifiedoligonucleotides (McGinnis et al 2019, Nature Methods). Oligonucleotidecell attachment creates the possibility of building cell barcodesdirectly on the cell, for example, by split pool barcode construction(Fan et al 2015, Science). These cell barcodes could, in turn, be usedto barcode the nucleic acid substrates from the targeted cell.

Previously, one requirement for this form of cell barcoding was that thecell, together with the complement of the attached cell barcodeoligonucleotides, had to be confined to a partition prior tooligonucleotide cleavage or release from the cell, at which pointattachment to the cellular nucleic acid substrate could occur. Althoughdroplets provide a partitioning format for this type of reaction, thereare some drawbacks. First, the upper limit to the number of droplets ina single emulsion is approximately 1-2 million. To minimize multiplecells co-localizing to single droplets, cells can be loaded at lambdasof approximately 0.05-0.1. Based on the number of droplets per emulsion,the number of cells encapsulated is limited to 50,000 to 100,000,maximally. This cell throughput may not be sufficient for some types ofexperiments and suffers from upwards scalability limits. Second, due tocell suspensions leftover in the inlet, the microfluidics and indroplets not harvested from the outlet, cell loss using dropletmicrofluidics is difficult to eliminate resulting in cell utilization ofapproximately 60-85%. For precious cells, this level of cell utilizationmay not be sufficient. Third, droplet microfluidics require chips, oils,and instruments, which are all expensive and difficult to support.Fourth, adding reagents to already formed droplets and washing theproducts while still maintaining droplets is not easily engineered,albeit feasible through picoinjection, droplet merging and magnetic beadtrapping approaches. This constraint makes single-cell DNA analysisdifficult since proteinase K digestion followed by inactivation and thenbiochemistry addition is not supported by simple droplet microfluidics.Fifth, since only 1-10 million oligonucleotides can be loaded onto cellswithout disrupting their membrane, droplets have to be tens of pL involume to provide sufficient oligo concentrations to drive molecularbiology reactions. These small droplets may be difficult to achieve withtwo aqueous inlet microfluidics. Sixth, cells usually have to be washedto remove their media prior to barcoding reactions. This contributessignificantly to cell loss.

The present methods solve the above limitations with droplets asfollows: Subsequent to building the cell barcodes on cells, the cellsare resuspended with hydrogel solution that is density matched for thecells such that cells do not settle. This can be achieved with commonreagents used to keep cells in suspension such as sucrose cushions,Percoll (Sigma) and/or Optiprep (Sigma). The solidification of thehydrogel then ensues. The mechanism behind solidification depends on thematerial used for the hydrogel. For example, agarose solidificationwould be caused by a drop in temperature. Alternatively, alginate can becrosslinked using calcium. Alternatively, TEMED initiates thecrosslinking of polyacrylamide monomers. The cells are thus dispersedthroughout the solidified hydrogel matrix.

The hydrogel may or not be modified to bind the cell barcodeoligonucleotide. For example, the cell barcode oligonucleotide can bemodified at the 5′ end with biotin, and an avidin analog such asstreptavidin, can be conjugated to the hydrogel material. Thus, when insolution, the cells with the bound oligonucleotides will move freely,however, once the hydrogel solidifies any released oligonucleotides willbind to the matrix in the direct vicinity of the cell membrane to form askin or shell where the cell membrane exists. Hydrogels that are between0.01 to 10% wt/vol for example are porous to ionic and non-ionicdetergents, as well as low molecular weight proteins, enzymes, andco-factors. Thus after trapping the cells in the hydrogel matrix, celllysis reagents (e.g., 0.1% NP-40) can be applied to the cells. The lysisreagents will diffuse through the matrix and lyse the cells. Cellbarcode oligonucleotide cleavage or release will occur either as adirect result of cell lysis and membrane dissolution or through aspecific or non-specific agent that cleaves or releases theoligonucleotide from the cell. The released cell-substrate nucleic acidswill then bind to the cell barcodes oligonucleotides that have beenimmobilized into a shell at the cell membrane/hydrogel interface.

The volume of the area encircled by the cell/hydrogel interface isessentially the volume of the cell. This minimal volume willsignificantly increase the effective concentration of the cell barcodeoligonucleotides to a maximum value whether they are immobilized to theshell of oligonucleotides or not. This can compensate, for example, forthe limited number of cell barcode oligonucleotides that can be loadedonto cells without affecting their physiology, e.g., 1-10 millionoligonucelotides per cell. For cells that are approximately 9 microns indiameter, the effective concentration with 2 million oligonucleotideswould be several hundred nM, which is a concentration sufficient tosupport most molecular biology reactions, such as reverse transcription.Low molecular weight RNA or DNA dependent polymerases can either beadded together with the lysis reagent or after and this may also besubsequent to an intervening wash to remove or inactivate the cell lysisreagent.

Once cell barcoding occurs as a result of cell barcode oligonucleotidetagging of the cellular substrate nucleic acid, the final steps oflibrary prep can occur either in the hydrogel matrix or in solutionafter removing the material from the hydrogel matrix that has or has notbeen solubilized. For example, reverse transcriptase, due to its lowmolecular weight size, will flow through hydrogels of up to 5% incomposition. Applying this with the lysis reagent together with orwithout an oligonucleotide cleavage/release agent would lead to thefollowing events. As the cell membrane dissolves with the lysis reagent,the cell barcode oligonucleotides will bind to the hydrogel to form ashell where the cell membrane existed. Released RNA will bind to theimmobilized oligonucleotides at the shell where the cell membraneexisted and reverse transcriptase will synthesize cDNA. This is thebarcoding reaction. Once this occurs, the hydrogel can be dissolved andfinal steps to prepare the NGS library can be done in bulk.

None of the above workflows require microfluidics (chips, oils, andinstruments) and occurs in bulk. One benefit of this format is thatmulti-step reactions can be supported. For example, if DNA genotypeinformation is wanted, the first reagent to flow through the hydrogelcould be proteinase K, for example, thermo-sensitive proteinase K. Thiswill digest the nucleosomes and chromatin accessory proteins leaving theDNA accessible to further molecular biology. Through the destruction ofthe cell membrane the cell barcode oligonucleotides can bind to form ashell where the cell membrane existed. Proteinase K can be deactivated,DNA polymerase together with reagents can flow into the hydrogel andbarcoding through template-directed DNA synthesis, for example, canoccur. Once barcoded, the final steps for library preparation can occureither inside or outside the hydrogel.

Cells in their native media can be mixed with the hydrogel material.Once solidified, the hydrogel can be washed to remove the media.Furthermore, since each cell will have a cell barcode clonal set ofoligonucleotides, each cell captured in the hydrogel matrix will bebarcoded. These two factors will increase cell utilization from thestarting material to close to 100%.

In some aspects, a mixture of individual cells or individual cell nucleiand cross-linked hydrogel is provided. In some embodiments, theindividual cells comprise heterologous oligonucleotides attached to cellmembranes of the individual cells or the individual cell nuclei compriseheterologous oligonucleotides attached to nuclear membranes of theindividual cell nuclei.

In some embodiments, the individual cells comprise heterologousoligonucleotides anchored in cell membranes of the individual cells orthe individual cell nuclei comprise heterologous oligonucleotidesanchored in nuclear membranes of the individual cell nuclei. In someembodiments, the heterologous barcoded oligonucleotides comprise a lipidmoiety and wherein the lipid moiety anchors the heterologous barcodedoligonucleotides in the cell membranes.

In some embodiments, the hydrogel is covalently linked to a moleculehaving binding affinity for the heterologous oligonucleotides. In someembodiments, the hydrogel is non-covalently linked to a molecule havingbinding affinity for the heterologous oligonucleotides. In someembodiments, the molecule is selected from the group consisting ofbiotin, streptavidin, an antibody, an aptamer, nickel (Ni), europium(Eu) or a polynucleotide comprising a sequence of at least 6 contiguousnucleotides that is fully complementary to a sequence in theheterologous barcoded oligonucleotides.

In some embodiments, the cells are mammalian cells. In some embodiments,the nuclei or cells comprise fragmented nuclear DNA, wherein thefragmented DNA comprises common adapter sequences at ends of thefragments.

In some embodiments, the hydrogel comprises alginate, agarose,polyacrylamide, chitosan, hyaluronan, dextran, collagen, fibrin,polyethylene glycol (PEG), poly(hydroxyethyl methacrylate) (polyHEMA),polyvinyl alcohol (PVA) or polycaprolactone (PCL).

In some embodiments, the heterologous barcoded oligonucleotides comprisea cell-specific barcode sequence and a 3′ sequence. In some embodiments,the 3′ sequence is a polyT sequence of at least 5 contiguous thymines.In some embodiments, the 3′ sequence is a random sequence of at least 5(e.g., at least 8, at least 10, at least 12, e.g., 6-30) contiguousnucleotides. In some embodiments, the 3′ sequence is a targetgene-specific sequence of at least 5 contiguous nucleotides. In someembodiments, the 3′ sequence is an adapter of at least 5 (e.g., 5-100,5-25) contiguous nucleotides. In some embodiments, the adapter can becomplementary to common adapter sequences at the end of fragmented DNAfrom the cell or nuclei for example.

In some embodiments, the heterologous barcoded oligonucleotides furthercomprise a 5′ PCR handle sequence.

In some aspects, a method of tagging cell-specific barcodes to cellnucleic acids is provided. In some embodiments, the method comprises:providing (i) cells or isolated cell nuclei having heterologous barcodedoligonucleotides attached to cell membranes of the cells or (ii) cellnuclei comprising heterologous oligonucleotides attached to nuclearmembranes of the individual cell nuclei; mixing the cells or nuclei witha liquid-form hydrogel; cross-linking the hydrogel around the cells ornuclei, wherein the hydrogel forms a solid gel; releasing theheterologous barcoded oligonucleotides from the cell membranes ornuclear membranes to generate released heterologous barcodedoligonucleotides; allowing the heterologous barcoded oligonucleotidesreleased from the cell membranes or nuclear membranes to locate atsolidified hydrogel surrounding the cells or nuclei; attaching theheterologous barcoded oligonucleotides to cell polynucleotides or copiesor cDNAs thereof to form barcoded cell polynucleotides; and dissolvingthe solidified hydrogel or extracting the barcoded cell polynucleotidesfrom the solidified hydrogel, thereby releasing barcoded cellpolynucleotides from the hydrogel, thereby tagging cell-specificbarcodes to cell nucleic acids.

In some embodiments, the allowing comprises binding the heterologousbarcoded oligonucleotides released from the cell membranes or nuclearmembranes to the solidified hydrogel surrounding the cells or nuclei. Insome embodiments, the allowing comprises diffusion of the heterologousbarcoded oligonucleotides released from the cell membranes or nuclearmembranes to the solidified hydrogel surrounding the cells or nucleisuch that the heterologous barcoded oligonucleotides are localized at ahydrogel/membrane interface. In some embodiments, the extracting thebarcoded cell polynucleotides from the solidified hydrogel compriseselectrophoresing the barcoded cell polynucleotides from the solidifiedhydrogel.

In some embodiments, the method further comprises sequencing thebarcoded cell polynucleotides released from the hydrogel.

In some embodiments, the heterologous barcoded oligonucleotides comprisea lipid moiety and wherein the lipid moiety anchors the heterologousbarcoded oligonucleotides in the cell membranes or nuclear membranes.

In some embodiments, the hydrogel is covalently linked to a moleculehaving binding affinity for the heterologous oligonucleotides. In someembodiments, the molecule is selected from the group consisting ofbiotin, streptavidin, an antibody, an aptamer, Ni, Eu, or apolynucleotide comprising a sequence of at least 6 contiguousnucleotides that is fully complementary to a sequence in theheterologous barcoded oligonucleotides.

In some embodiments, the cells are mammalian cells.

In some embodiments, the nuclei or cells comprise fragmented nuclearDNA, wherein the fragmented DNA comprises common adapter sequences atends of the fragments. In some embodiments, the method further comprisesfragmenting the nuclear DNA and introducing the common adapter sequencewith a transposase (e.g., via tagmentation).

In some embodiments, the hydrogel comprises alginate, agarose,polyacrylamide, chitosan, hyaluronan, dextran, collagen, fibrin,polyethylene glycol (PEG), poly(hydroxyethyl methacrylate) (polyHEMA),polyvinyl alcohol (PVA) or polycaprolactone (PCL). In some embodiments,the hydrogel comprises alginate and the crosslinking comprisescontacting the hydrogel with calcium. In some embodiments, the hydrogelis covalently linked to a molecule having binding affinity for theheterologous barcoded oligonucleotides and the released heterologousbarcoded oligonucleotides bind to the molecule at a hydrogel/cellinterface or hydrogel/nuclei interface. In some embodiments, themolecule is streptavidin and the heterologous barcoded oligonucleotidesbiotinylated. In some embodiments, the molecule is a polynucleotidecomprising a sequence of at least 6 contiguous nucleotides that is fullycomplementary to a sequence in the heterologous barcodedoligonucleotides such that the polynucleotide linked to the hydrogelhybridizes to the released heterologous barcoded oligonucleotides.

In some embodiments, the releasing comprises lysing the cells or nuclei.In some embodiments, the lysing comprising contacting the cells ornuclei in the hydrogel with an ionic or non-ionic detergent. In someembodiments, the lysing comprising contacting the cells to a protease(for example, proteinase K).

In some embodiments, the releasing comprises cleaving the heterologousbarcoded oligonucleotides from a portion of the oligonucleotides to freethe heterologous barcoded oligonucleotides from the cell membrane ornuclear membrane.

In some embodiments, the attaching comprises ligating the heterologousbarcoded oligonucleotides to cell polynucleotides or copies or cDNAsthereof to form the barcoded cell polynucleotides.

In some embodiments, the attaching comprises hybridizing at least the 3′end of the heterologous barcoded oligonucleotides to cellpolynucleotides or copies or cDNAs thereof. In some embodiments, themethod further comprises extending the 3′ end in a template-specificmanner with a polymerase to form the barcoded cell polynucleotides.

In some embodiments, after the lysing and before the attaching themethod comprises contacting the cells with a reverse transcriptase underconditions to form cDNAs from RNA in the cells; and the attachingcomprises attaching the heterologous barcoded oligonucleotides to thecDNAs.

In some embodiments, the heterologous barcoded oligonucleotides comprisebarcode sequences that are unique for the cells or nuclei to which theheterologous barcoded oligonucleotides are attached.

In some embodiments, the providing comprises synthesizing theheterologous barcoded oligonucleotides on the cells or nuclei using asplit and pool methodology such that different cells or different nucleiare linked to a plurality of identical heterologous barcodedoligonucleotides and where different cells or different nuclei haveunique heterologous barcoded oligonucleotides.

In some embodiments, the heterologous barcoded oligonucleotides comprisea cell-specific barcode sequence and a 3′ sequence. In some embodiments,the 3′ sequence is a polyT sequence of at least 5 contiguous thymines.In some embodiments, the 3′ sequence is a random sequence of at least 5contiguous nucleotides. In some embodiments, the 3′ sequence is a targetgene-specific sequence of at least 5 (e.g., at least 8, at least 10, atleast 12, e.g., 6-30) contiguous nucleotides. In some embodiments, the3′ sequence is an adapter of at least 5 (e.g., 5-100, 5-25) contiguousnucleotides. In some embodiments, the adapter can be complementary tocommon adapter sequences at the end of fragmented DNA from the cell ornuclei for example. In some embodiments, the heterologous barcodedoligonucleotides further comprise a 5′ PCR handle sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a lipid-modified oligonucleotide comprising a cell orsample barcode linked to a cell. An acrydite moiety is present on the 3′end of the anchor oligonucleotide. FIG. 1 discloses“GTAACGATCCAGCTGTCACTTGGAATTCTCGGGTGCCAAGG” as SEQ ID NO: 6,“CCTTGGCACCCGAGAATTCCA” as SEQ ID NO: 7, and “AGTGACAGCTGGATCGTTAC” asSEQ ID NO: 8.

FIG. 2 depicts a lipid-modified oligonucleotide comprising a cell orsample barcode linked to a cell. Imidazole triphosphate nucleosides arepresent on the 3′ end of the anchor oligonucleotide. FIG. 2 discloses“GTAACGATCCAGCTGTCACTTGGAATTCTCGGGTGCCAAGG” as SEQ ID NO: 6,“CCTTGGCACCCGAGAATTCCA” as SEQ ID NO: 7, and “AGTGACAGCTGGATCGTTAC” asSEQ ID NO: 8.

FIG. 3A-B depicts an exemplary workflow. FIG. 3B continues from FIG. 3A.

DEFINITIONS

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a bead” includes a plurality of such beads andreference to “the sequence” includes reference to one or more sequencesknown to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2nd ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well-known and commonlyemployed in the art.

The term “barcode” refers to a short nucleotide sequence (e.g., at leastabout 2, 3, 4, 6, 8, 10, 12, 14, 16, 20, 25 or more (e.g., 4-30, 5-25,5-20) nucleotides long) that identifies a molecule to which it isconjugated. Barcodes can be used, e.g., to identify molecules in a cellor nucleus. Such a cell-specific or nucleus-specific barcode should beunique for that cell or nucleus as compared to barcodes present in othercells or nuclei. Once associated with a cell-specific ornucleus-specific barcode, nucleic acids from each cell can bedistinguished from nucleic acid of other cells due to the uniquebarcode. In some cases, the cell-specific or nucleus-specific barcode isgenerated using a split and mix (also referred to as split and pool)synthetic scheme.

Additional types of barcodes can also be included in a polynucleotidehaving a cell-specific or nucleus-specific barcode. For example,additional barcodes can uniquely identify the molecule to which it isconjugated. Such barcodes are useful for determining the number oforiginal molecule sin a sample for instance.

The length of the barcode sequence determines how many unique samplescan be differentiated. For example, a 1 nucleotide barcode candifferentiate 4, or fewer, different samples or molecules; a 4nucleotide barcode can differentiate 4⁴ or 256 samples or less; a 6nucleotide barcode can differentiate 4096 different samples or less; andan 8 nucleotide barcode can index 65,536 different samples or less.

Barcodes can be synthesized and/or polymerized (e.g., amplified) usingprocesses that are inherently inexact. Thus, barcodes that are meant tobe uniform (e.g., a cell-specific barcode shared amongst all barcodednucleic acid of a cell) can contain various N−1 deletions or othermutations from the canonical barcode sequence. Thus, barcodes that arereferred to as “identical or substantially identical copies” can includebarcodes that differ due to one or more errors in, e.g., synthesis,polymerization, or purification and thus contain various N−1 deletionsor other mutations from the canonical barcode sequence. Moreover, therandom conjugation of barcode nucleotides during synthesis using e.g., asplit and pool approach and/or an equal mixture of nucleotide precursormolecules as described herein, can lead to low probability events inwhich a barcode is not absolutely unique (e.g., different from otherbarcodes of a population or different from barcodes of a differentpartition, cell, or bead). However, such minor variations fromtheoretically ideal barcodes do not interfere with the single cellanalysis methods, compositions, and kits described herein. Therefore, asused herein, the term “unique” in the context of a particle, cellular,partition-specific, or molecular barcode encompasses various inadvertentN−1 deletions and mutations from the ideal barcode sequence. In somecases, issues due to the inexact nature of barcode synthesis,polymerization, and/or amplification, are overcome by oversampling ofpossible barcode sequences as compared to the number of barcodesequences to be distinguished (e.g., at least about 2-, 5-, 10-fold ormore possible barcode sequences). For example, 10,000 cells can beanalyzed using a cellular barcode having 9 barcode nucleotides,representing 262,144 possible barcode sequences. The use of barcodetechnology is known in the art, see for example Katsuyuki Shiroguchi, etal. Proc Natl Acad Sci USA., 2012 Jan. 24; 109(4):1347-52; and Smith, AM et al., Nucleic Acids Research (2010), 38(13):e142.

The term “amplification reaction” refers to any in vitro method formultiplying the copies of a target sequence of nucleic acid in a linearor exponential manner. Such methods include, but are not limited to,polymerase chain reaction (PCR); DNA ligase chain reaction (LCR); QBetaRNA replicase and RNA transcription-based amplification reactions (e.g.,amplification that involves T7, T3, or SP6 primed RNA polymerization),such as the transcription amplification system (TAS), nucleic acidsequence based amplification (NASBA), and self-sustained sequencereplication (3 SR); single-primer isothermal amplification (SPIA), loopmediated isothermal amplification (LAMP), strand displacementamplification (SDA); multiple displacement amplification (MDA); rollingcircle amplification (RCA); as well as others known to those of skill inthe art. See, e.g., Fakruddin et al., J. Pharm Bioallied Sci. 20135(4):245-252.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, such as is obtained with cyclesequencing or linear amplification.

A nucleic acid, or portion thereof, “hybridizes” to another nucleic acidunder conditions such that non-specific hybridization is minimal at adefined temperature in a physiological buffer. In some cases, a nucleicacid, or portion thereof, hybridizes to a conserved sequence sharedamong a group of target nucleic acids. In some cases, a primer, orportion thereof, can hybridize to a primer binding site if there are atleast about 6, 8, 10, 12, 14, 16, or 18 contiguous complementarynucleotides, including “universal” nucleotides that are complementary tomore than one nucleotide partner. Alternatively, a primer, or portionthereof, can hybridize to a primer binding site if there are fewer than1 or 2 complementarity mismatches over at least about 12, 14, 16, or 18contiguous complementary nucleotides. In some embodiments, the definedtemperature at which specific hybridization occurs is room temperature.In some embodiments, the defined temperature at which specifichybridization occurs is higher than room temperature. In someembodiments, the defined temperature at which specific hybridizationoccurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C.

The term “oligonucleotide” is not intended to be limited to a specificnumber of nucleotides. In some embodiments, an oligonucleotide can have10-500 nucleotides, e.g., 20-200 or 15-100 nucleotides in length.

The term “partitioning” or “partitioned” refers to separating a sampleinto a plurality of portions, or “partitions.” Partitions are generallyphysical, such that a sample in one partition does not, or does notsubstantially, mix with a sample in an adjacent partition. Partitionscan be solid or fluid. In some embodiments, a partition is a solidpartition, e.g., a microchannel, well, tube, and plate. In someembodiments, a partition is a fluid partition, e.g., a droplet. In someembodiments, a fluid partition (e.g., a droplet) is a mixture ofimmiscible fluids (e.g., water and oil). In some embodiments, a fluidpartition (e.g., a droplet) is an aqueous droplet that is surrounded byan immiscible carrier fluid (e.g., oil).

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof.

Modifications include, but are not limited to, those providing chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, points of attachment andfunctionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,peptide nucleic acids (PNAs), phosphodiester group modifications (e.g.,phosphorothioates, methylphosphonates), 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Nucleic acids can alsoinclude non-natural bases, such as, for example, nitroindole.Modifications can also include 3′ and 5′ modifications including but notlimited to capping with a fluorophore (e.g., quantum dot) or anothermoiety.

A nucleic acid, or a portion thereof, “hybridizes” to another nucleicacid under conditions such that non-specific hybridization is minimal ata defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mMchloride salt). In some cases, a nucleic acid, or portion thereof,hybridizes to a conserved sequence shared among a group of targetnucleic acids. In some cases, a primer, or portion thereof, canhybridize to a primer binding site if there are at least about 6, 8, 10,12, 14, 16, or 18 contiguous complementary nucleotides, including“universal” nucleotides that are complementary to more than onenucleotide partner. Alternatively, a primer, or portion thereof, canhybridize to a primer binding site if there are fewer than 1 or 2complementarity mismatches over at least about 12, 14, 16, or 18contiguous complementary nucleotides. In some embodiments, the definedtemperature at which specific hybridization occurs is room temperature.In some embodiments, the defined temperature at which specifichybridization occurs is higher than room temperature. In someembodiments, the defined temperature at which specific hybridizationoccurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C. In some embodiments, the defined temperature at which specifichybridization occurs is 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C.

The term “primer” refers to a polynucleotide sequence that hybridizes toa sequence on a target nucleic acid and serves as a point of initiationof nucleic acid synthesis. Primers can be of a variety of lengths andare often less than 50 nucleotides in length, for example 12-30nucleotides, in length. The length and sequences of primers for use inPCR can be designed based on principles known to those of skill in theart, see, e.g., PCR Protocols: A Guide to Methods and Applications,Innis et al., eds, 1990. Primers can be DNA, RNA, or a chimera of DNAand RNA portions. In some cases, primers can include one or moremodified or non-natural nucleotide bases. In some cases, primers arelabeled.

The term “target nucleic acid” refers to a polynucleotide such as DNA,e.g., single stranded DNA or double stranded DNA, RNA, e.g., mRNA ormiRNA, or a DNA-RNA hybrid. DNA includes genomic DNA and complementaryDNA (cDNA).

As used herein, the term “heterologous” refers to two components (e.g.,a cell and a barcode oligonucleotide) that are not found together innature, e.g., because they are not found together in the same wild-typeorganism.

The term “template nucleic acid” refers to a polynucleotide templatethat is used to generate a second polynucleotide strand that can becomplementary to the template or a portion thereof. In some embodiments,in a reverse transcription reaction an RNA template is used to generatea DNA that is complementary to the RNA. In other embodiments, a firststrand cDNA is used as a template during polymerase based amplificationto generate a second stand cDNA that is complementary to the firststrand.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered a new way of associating polynucleotidebarcodes with individuals cell nucleic acids. An advantage of themethods described herein is that individual cells do not need to beinserted into droplets or other partitions, which can lead toinefficiencies of matching single cells with single partitions or otherissues as described herein. Instead, embodiments described herein caninvolve forming a solid hydrogel around a cell mixture such that cellsare separated from each other by the hydrogel, wherein the cells havecell-specific barcode oligonucleotide attached to cell membranes.Similarly, cell nuclei can alternatively be used to form a solidhydrogel with isolated nuclei separated from each other, wherein thenuclei have nuclei-specific barcode oligonucleotide attached to nuclearmembranes. Regardless of whether individual cells or nuclei are used,once the cells or nuclei are embedded in the solid hydrogel, the barcodeoligonucleotides can be released from the membranes. Because the cellsor nuclei are embedded in the hydrogel, the barcode oligonucleotideswill not diffuse beyond the interface of the cell or nucleus and thehydrogel. In some embodiments, the hydrogel can include a moleculehaving affinity for the barcode oligonucleotides, further maintainingthe position of the barcode oligonucleotides. The cells or nuclei canthen be lysed or permeabilized to allow nucleic acids (DNA, RNA orcopies thereof) from the cells or nuclei to associate with the barcodeoligonucleotides at the interface of the cell or nucleus and thehydrogel. The cell-specific or nucleus-specific barcode oligonucleotideis associated with the cell or nucleus nucleic acid, thereby barcodingthe nucleic acids and allowing for subsequent mixing of the nucleicacids from the different cells or nuclei for analysis without losingtrack of the cell or nuclear origin of individual nucleic acids. Thisand other aspects are described herein.

Any type of cells can be used according to the methods and compositionsdescribed herein. In some embodiments, the cells are mammalian, forexample human cells. In some embodiments, the cells are from abiological sample. Biological samples can be obtained from anybiological organism, e.g., an animal, plant, fungus, pathogen (e.g.,bacteria or virus), or any other organism. In some embodiments, thebiological sample is from an animal, e.g., a mammal (e.g., a human or anon-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), abird (e.g., chicken), or a fish. A biological sample can be any tissueor bodily fluid obtained from the biological organism, e.g., blood, ablood fraction, or a blood product (e.g., serum, plasma, platelets, redblood cells, and the like), sputum or saliva, tissue (e.g., kidney,lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletalmuscle, cartilage, or bone tissue); cultured cells, e.g., primarycultures, explants, and transformed cells, stem cells, or cells found instool, urine, etc.

In some embodiments, isolated nuclei are provided. Methods of formingisolated nuclei are known and can be used as desired. Exemplary methodsof generating isolated nuclei include those described in U.S. Pat. No.8,546,134; Gaublomme, et al., Nature Communications volume 10, Articlenumber: 2907 (2019). In some embodiments, the nuclei comprise fragmentednuclear DNA, allowing for example, for a variation of ATAC-seq. Forexample, the cells can be permeabilized and the nuclear DNA within canbe fragmented, for example with a tranposase that introduces adaptersequences to the ends of the fragmented DNA. Where isolated nuclei areused, the nuclei need not be permeabilized for entry to the transposaseinto the nuclei. The action of the transposase sometimes referred to as“tagmentation” and can involve introduction of different adaptersequences on different sides of a DNA breakage point or the adaptersequences added can be identical. In either case, the adapter sequencesare common adapter sequences in that the adapter sequences are the sameacross a diversity of DNA fragments. Homoadapter-loaded tagmentases aretagmentases that contain adapters of only one sequence, which adapter isadded to both ends of a tagmentase-induced breakpoint in the genomicDNA. Heteroadapter-loaded tagmentases are tagmentases that contain twodifferent adapters, such that a different adapter sequence is added tothe two DNA ends created by a tagmentase-induced breakpoint in the DNA.Adapter loaded tagmentases are further described, e.g., in U.S. PatentPublication Nos: 2010/0120098; 2012/0301925; and 2015/0291942 and U.S.Pat. Nos. 5,965,443; 6,437,109; 7,083,980; 9,005,935; and 9,238,671, thecontents of each of which are hereby incorporated by reference in theentirety for all purposes. By quantifying the number of intactsequencing reads (indicating heterochromatin not cleaved by transposase)one can measure chromatin structure.

A tagmentase is an enzyme that is capable of forming a functionalcomplex with a transposon end-containing composition and catalyzinginsertion or transposition of the transposon end-containing compositioninto the double-stranded target DNA with which it is incubated in an invitro transposition reaction. Exemplary transposases include but are notlimited to modified Tn5 transposases that are hyperactive compared towildtype Tn5, for example can have one or more mutations selected fromE54K, M56A, or L372P. Wild-type Tn5 transposon is a composite transposonin which two near-identical insertion sequences (IS50L and IS50R) areflanking three antibiotic resistance genes (Reznikoff W S. Annu RevGenet 42: 269-286 (2008)). Each IS50 contains two inverted 19-bp endsequences (ESs), an outside end (OE) and an inside end (IE). However,wild-type ESs have a relatively low activity and were replaced in vitroby hyperactive mosaic end (ME) sequences. A complex of the transposasewith the 19-bp ME is thus all that is necessary for transposition tooccur, provided that the intervening DNA is long enough to bring two ofthese sequences close together to form an active Tn5 transposasehomodimer (Reznikoff W S., Mol Microbiol 47: 1199-1206 (2003)).Transposition is a very infrequent event in vivo, and hyperactivemutants were historically derived by introducing three missensemutations in the 476 residues of the Tn5 protein (E54K, M56A, L372P),which is encoded by IS50R (Goryshin I Y, Reznikoff W S. 1998. J BiolChem 273: 7367-7374 (1998)). Transposition works through a“cut-and-paste” mechanism, where the Tn5 excises itself from the donorDNA and inserts into a target sequence, creating a 9-bp duplication ofthe target (Schaller H. Cold Spring Harb Symp Quant Biol 43: 401-408(1979); Reznikoff W S., Annu Rev Genet 42: 269-286 (2008)). In currentcommercial solutions (Nextera™ DNA kits, Illumina), free synthetic MEadapters are end-joined to the 5′-end of the target DNA by thetransposase (tagmentase). In some embodiments, the tagmentase is linkedto a solid support (e.g., a bead that is different from the bead linkedto the forward primer). An example commercial bead-linked tagmentase isNextera™ DNA Flex (Illumina).

In some embodiments, the adapter(s) is at least 19 nucleotides inlength, e.g., 19-100 nucleotides. In some embodiments, the adapters aredouble stranded with a 5′ end overhang, wherein the 5′ overhand sequenceis different between heteroadapters, while the double stranded portion(typically 19 bp) is the same. In some embodiments, an adapter comprisesTCGTCGGCAGCGTC (SEQ ID NO:1) or GTCTCGTGGGCTCGG (SEQ ID NO:2). In someembodiments involving the heteroadapter-loaded tagmentase, thetagmentase is loaded with a first adapter comprising TCGTCGGCAGCGTC (SEQID NO:1) and a second adapter comprising GTCTCGTGGGCTCGG (SEQ ID NO:2).In some embodiments, the adapter comprises AGATGTGTATAAGAGACAG (SEQ IDNO:3) and the complement thereof (this is the mosaic end and this is theonly specifically required cis active sequence for Tn5 transposition).In some embodiments, the adapter comprisesTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO:4) with the complement forAGATGTGTATAAGAGACAG (SEQ ID NO:3) or GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ ID NO:5) with the complement for AGATGTGTATAAGAGACAG (SEQ ID NO:3).In some embodiments involving the heteroadapter-loaded tagmentase, thetagmentase is loaded with a first adapter comprisingTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO:4) with the complement forAGATGTGTATAAGAGACAG (SEQ ID NO:3) and GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ ID NO:5) with the complement for AGATGTGTATAAGAGACAG (SEQ ID NO:3).

Cells or isolated nuclei can be barcoded by attaching an oligonucleotidecomprising one or more barcode sequence to the cell membrane, or in thecase of isolated nuclei, to the nuclear membrane. Oligonucleotides canbe attached to cell or nuclear membranes in several ways. In someembodiments, lipid (LMO) or cholesterol (CMO)-modified oligonucleotidescan be mixed with the cells or isolated nuclei, allowing the lipid orcholesterol to embed in the membranes. For example, in some embodiments,an anchor, co-anchor and barcode oligonucleotide are hybridized insolution (FIGS. 1 and 2 depict an example). The cholesterol orlipid-derivative ends of the anchor and co-anchor embed into the cell ornuclear membrane. Protocols for barcoding cells using LMOs or CMOs aredescribed in, e.g., McGinnis, et al., Nature Methods 16:619-626 (2019);Weber et al., Biomacromolecules 15:4621-4626 (2014). In someembodiments, the LMOs comprise a spacer between the lipid moiety and thenucleic acid. The lipid moiety, in some embodiments, includes a longalkyl chain of 12-24 carbon atoms, e.g., 12-22, 12-20, 12-18, 14-22,14-20, 14-18, 16-22, 16-20, or 16-18 carbon atoms. The spacer may be forexample 10-80 nucleotides long, e.g., 10-60, 10-40, 20-80, 20-60, 20-40,40-60, 40-80, 50-80, 50-80, or 60-80 nucleotides long.

In some embodiments the barcode oligonucleotide is hybridized to anoligonucleotide embedded in the cell or nuclear membrane. For example anLMO or CMO embedded in a membrane can comprise a binding sequence (forexample 6-20 nucleotides in length) and an oligonucleotide comprising abarcode sequence can be hybridized to the binding sequence via acomplementary sequence in the barcode oligonucleotide. In someembodiments, the 3′ end of the barcode oligonucleotide is free to bindto the complementary cellular nucleic acid.

In yet another embodiment, streptavidin or other avidin analog isattached to the membrane (e.g., via lipid or cholesterol attachment) andthe barcode oligonucleotides are biotinylated allowing for binding ofthe barcode oligonucleotides to be attached to the streptavidin attachedto the membrane. In other embodiments, other affinity molecules can belinked to the barcode oligonucleotides such that the affinity moleculesbind to the cell or nuclear membrane or components or proteins therein.Examples of binding molecules include but are not limited to an antibodyor an aptamer. See, e.g., Stoekius, et al., Genome Biology 19:224(2018); Delley, et al., bioRxiv 1-10 (2017). In some embodiments, thebarcode oligonucleotides can be conjugated to the cells. See, e.g.,Gehring, et al., BioRxiv 1-19 (2018). The above options for attachingbarcodes to cells are intended as examples and are provided withoutlimitation.

In some embodiments, the barcode oligonucleotides further comprise anacrydite phosphoramidite moiety at the 3′ end of the oligonucleotides.See, e.g., Rehman, et al., Nuc. Acids Res. 27(20) 649-655 (1999).

In some embodiments, the barcode oligonucleotides further compriseimidazole triphosphate nucleosides at the 3′ end of theoligonucleotides. See, e.g. Rothlisberger, et al., ChemicalCommunications 53 13031-13034.

Cell specific barcodes can be synthesized on cells or nuclei for exampleusing a split and pool method. For example, an oligonucleotidecomprising a common sequence can be attached to cell or nuclearmembranes or cells or nuclei, respectively, to form a mixture of cellsor nuclei having the oligonucleotide in attached to the membranes. Themixture can then be split into portions, where each portion receives adifferent nucleotide added to the oligonucleotide. The cells or nucleiare then combined, mixed, and split into portions again. This process ifrepeated, resulting in a unique, cell-specific (or nuclei-specific)nucleotide sequence on the cells or nuclei. An example of split-and-poolmethods is provided in Fan, et al., Science 2015 Feb. 6;347(6222):1258367. Optionally a common capture sequence can be added tothe 3′ end of the oligonucleotides, such that the resultingoligonucleotides include a 5′ common sequence (optionally usable as aPCR handle, a cell-specific barcode, and a 3′ capture sequence.

While the description here describes cell-specific barcodes it will beappreciated that other types of barcodes can equally be used wherebarcodes are described herein. For example, in some embodiments, cellsfrom the same sample are all labelled with the same barcodeoligonucleotide sequence, but cells from different samples receivedifferent barcodes, thereby allowing for barcoding by sample rather thanby cell.

The 5′ common sequence can be selected as desired and can have variouslengths. In some embodiments, the 5′ common sequence has between 4-50nucleotides in length. The barcode sequence itself can vary in length.In some embodiments, the barcode is between 5-50 or 5-75 nucleotides inlength. A 3′ capture sequence can vary depending on the sequences to becaptured. In some embodiments, the capture sequence is a randomsequence, e.g., a random sequence or 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20or more (e.g., 2-50, 2-25, 5-30) nucleotides. In some embodiments, thecapture sequence is a homo-polymeric sequence (e.g., a polyA or a polyTsequence) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more (e.g., 2-50,2-25, 5-30) nucleotides. In some embodiments, the capture sequencecomprises a gene or target-specific capture sequence, for example of 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more (e.g., 2-50, 2-25, 5-30)nucleotides. In some embodiments, different barcoded oligonucleotides onthe same cell can have different 3′ capture sequences. This can beachieved for example by adding a mixture of 3′ capture sequences to theoligonucleotides on the cells or nuclei.

A hydrogel is a network of polymer chains that are hydrophilic,sometimes found as a colloidal gel in which water is the dispersionmedium. A three-dimensional solid results from the hydrophilic polymerchains being held together by cross-links. Cells or nuclei with barcodeoligonucleotides attached can be mixed with a non-cross-linked (e.g.,liquid) form of a hydrogel and then solidified (e.g., cross-linked).Exemplary hydrogels include but are not limited to those comprisingalginate, agarose, nickel-nitrilotriacetic acid (Ni-NTA) agarose (whichwill bind dIMTP nucleosides), europium-nitrilotriacetic acid (Eu-NTA)agarose (which will bind dIMTP nucleosides), polyacrylamide, chitosan,hyaluronan, dextran, collagen, fibrin, polyethylene glycol (PEG),poly(hydroxyethyl methacrylate) (polyHEMA), polyvinyl alcohol (PVA) orpolycaprolactone (PCL). Concentrations of the hydrogel can be selectedto provide a solid hydrogel to suspend the target cells. In someembodiments, the hydrogel concentration is between for example 0.1%-20%,e.g., 0.1-1, 0.1-10, 1-20, or 1-10% wt/vol. Cell concentration in thehydrogel can also be selected as desired. In some embodiment, the cellconcentration is 10 cells/mL, 100 cells/mL, 1000 cells/mL, 10 000cells/mL, 100 000 cells/mL, 1 000 000 cells/mL, 10 000 000 cells/mL, 100000 000 cells/mL and 1 000 000 000 cells/mL or ranges between any two ofthe listed values (e.g., 10-100 000 000 cells/mL).

In some aspects, the hydrogel comprises polyacrylamide and the barcodeoligonucleotides comprise a 3′ acrydite phosphoramidite moiety, allowingfor later linking of the moiety to the solidified acrylamide. Thisallows for immobilization of the barcode oligonucleotides at the cellmembrane (or nuclear membrane)/hydrogel interface. See, e.g., Rehman, etal., Nuc. Acids Res. 27(20) 649-655 (1999).

In other embodiments, the hydrogel can be linked to a molecule havingbinding affinity for the barcode oligonucleotides. These can be used tobetter anchor the barcode oligonucleotides to the hydrogel/cellinterface once the oligonucleotides are released from the cell. In someembodiments, the molecule has affinity to a nucleic acid sequence in theoligonucleotide. In some embodiments, the molecule has affinity to anaffinity partner moiety linked to the oligonucleotide. Exemplarymolecules having binding affinity include but are not limited to biotin,streptavidin, an antibody, an aptamer or a polynucleotide comprising asequence of at least 6 (e.g., at least 8, 10, 12, 15, 20, e.g., 6-20 ormore) contiguous nucleotides that is fully complementary to a sequencein the heterologous barcoded oligonucleotides. In embodiments where themolecule is biotin the barcode oligonucleotide is attached tostreptavidin (or other avidin analog). In embodiments where the moleculeis streptavidin the barcode oligonucleotide is attached to biotin. Inembodiments where the molecule is an antibody or an aptamer, the barcodeoligonucleotide will be linked to a moiety (e.g., a protein ornon-protein antigen) to which the antibody or aptamer specificallybinds.

Once the cells are embedded in the solidified hydrogel, hydrogelcross-linkage is initiated to form a solid hydrogel surrounding thecells. Initiating solidification of the hydrogel will depend on the typeof hydrogel used. Generally methods of initiating cross-linkage ofhydrogels is known. For example, agarose can be cross-linked whentreated with calcium. Polyacrylamide can be polymerized withcross-linkers such as N,N′-Bis(acryloyl)cystamine and the reaction canbe initiated by contacting the hydrogel with TEMED and ammoniumpersulfate (APS).

After the cells are embedded in the solidified hydrogel, the barcodeoligonucleotides attached to the cells can be released. Because thehydrogel is solidified around the cells, the released oligonucleotidesshould not diffuse very far from the membrane/hydrogel interface.Indeed, in embodiments where the hydrogel comprises molecules withaffinity to the oligonucleotides, diffusion should be further reducedthereby localizing the oligonucleotides at or near the cellmembrane/hydrogel interface.

Release of the oligonucleotide from the cells can occur in any number ofways. In some embodiments, the oligonucleotides are cleaved from thecell membranes. Cleavage can occur by contacting the oligonucleotideswith an enzyme that cleaves the oligonucleotides from the cells, e.g.,in a sequence-specific manner of by cleaving a moiety that is part ofthe linkage of the oligonucleotide to the cell membrane.

In some embodiments, the release of the oligonucleotides is achieved bylysis of the cells. Lysis can occur by, for example, introduction of oneor more reagents into the hydrogel to achieve lysis. Exemplary reagentscan include, for example, an ionic or non-ionic detergent, a protease(e.g., proteinase K), or both.

In some embodiments, release of the barcodes is achieved without lysingthe cells or nuclei. In some of these embodiments, intact cells ornuclei can be permeabilized to allow entry of reagents. Exemplaryreagents can include the use of digitonin, or fixatives such asmethanol, or paraformaldehyde.

Once released from the cells or nuclei, the barcode oligonucleotides areattached covalently or non-covalently to cellular or nuclearpolynucleotides (e.g., genomic DNA, mRNA, small RNAs) or copies thereof(e.g., cDNAs). In embodiments in which the barcode oligonucleotidescomprise a 3′ capture sequence, cellular or nuclear polynucleotides orcopies thereof can be hybridized to the barcode oligonucleotides and apolymerase can be added to the hydrogel to extend the barcodeoligonucleotide in a template-dependent manner using the cellularpolynucleotides as templates. In some embodiments, the 3′ capturesequence is a polyT sequence and a reverse-transcriptase is used to forma first-strand cDNA from cellular RNA. In other embodiments, the lysedcells have been contacted with a reverse transcriptase and primer toform a first strand cDNA and then the barcode oligonucleotide isextended using the first strand cDNA as a template. In some embodiments,the cellular or nuclear polynucleotides are hybridized to the cellularbarcodes and thus the barcodes and the cellular polynucleotides arelinked at this point non-covalently. In other embodiments, the barcodeoligonucleotides can be ligated to cellular or nuclear polynucleotideseither directly or following enzymatic cleavage and/or polishing of endsof the cellular polynucleotides. In any case, because the barcodedoligonucleotides are localized to the cells from which they werereleased, the barcode oligonucleotides will be attached topolynucleotides or copies thereof of the cell or nucleus from which theywere released. This allows for cell-specific barcoding of a cell's ornucleus's polynucleotides. Because a number of cells or nuclei arepresent in the hydrogel, this occurs in parallel in each cell or nucleusin the hydrogel without the formation of partitions (e.g., withoutformation of droplets, microfluidic channels, microwells, etc.).Moreover, a 1:1 ratio of barcode to cell is readily achieved in themethods described herein achieved, in contrast to the difficulties thatcan occur when attempting 1:1 delivery of different reagents, cells,etc., into partitions.

Once the cellular (or nuclear) polynucleotides are associated with thebarcode oligonucleotides, the resulting barcoded cell polynucleotidescan be released from the solidified hydrogel, for example by extraction(for example by electrophoresis) or melting of the hydrogel. In someembodiments, the hydrogel can be melted, thereby releasing and poolingthe barcoded cellular polynucleotides. The hydrogel melting can beachieved as desired so long as the attachment of the barcodeoligonucleotide to the cellular or nuclear polynucleotide is notdisrupted. Melting conditions can include for example raising thetemperature or contacting the hydrogel with one or more reagents thatdepolymerizes the gel. Exemplary depolymerizing reagents can includereducing agents such as dithiothreitol (DTT) orTris(2-carboxyethyl)phosphine (TCEP).

One can subsequently analyze the resulting pool of barcodedpolynucleotides as desired. In some embodiments, the polynucleotides canbe nucleotide sequenced. By detecting the barcode sequence associatedwith the linked polynucleotide, one can determine from which cell eachsequencing read came from. In some embodiments, one can sort sequencingreads by their barcode. For example, one can determine the relativeamounts of a gene product in different cells, where different cells geneproducts are identified by having different barcodes.

Methods for high throughput sequencing and genotyping are known in theart. For example, such sequencing technologies include, but are notlimited to, pyrosequencing, sequencing-by-ligation, single moleculesequencing, sequence-by-synthesis (SBS), massive parallel clonal,massive parallel single molecule SBS, massive parallel single moleculereal-time, massive parallel single molecule real-time nanoporetechnology, etc. Morozova and Marra provide a review of some suchtechnologies in Genomics, 92: 255 (2008), herein incorporated byreference in its entirety.

Exemplary DNA sequencing techniques include fluorescence-basedsequencing methodologies (See, e.g., Birren et al., Genome Analysis:Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated byreference in its entirety). In some embodiments, automated sequencingtechniques understood in that art are utilized. In some embodiments, thepresent technology provides parallel sequencing of partitioned amplicons(PCT Publication No. WO 2006/084,132, herein incorporated by referencein its entirety). In some embodiments, DNA sequencing is achieved byparallel oligonucleotide extension (See, e.g., U.S. Pat. Nos. 5,750,341;and 6,306,597, both of which are herein incorporated by reference intheir entireties). Additional examples of sequencing techniques includethe Church polony technology (Mitra et al., 2003, AnalyticalBiochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732;and U.S. Pat. Nos. 6,432,360; 6,485,944; 6,511,803; herein incorporatedby reference in their entireties), the 454 picotiter pyrosequencingtechnology (Margulies et al., 2005 Nature 437, 376-380; U.S. PublicationNo. 2005/0130173; herein incorporated by reference in their entireties),the Solexa single base addition technology (Bennett et al., 2005,Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833,246;herein incorporated by reference in their entireties), the Lynxmassively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5,695,934;5,714,330; herein incorporated by reference in their entireties), andthe Adessi PCR colony technology (Adessi et al. (2000). Nucleic AcidRes. 28, E87; WO 2000/018957; herein incorporated by reference in itsentirety).

Typically, high throughput sequencing methods share the common featureof massively parallel, high-throughput strategies, with the goal oflower costs in comparison to older sequencing methods (See, e.g.,Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al.,Nature Rev. Microbiol., 7:287-296; each herein incorporated by referencein their entirety). Such methods can be broadly divided into those thattypically use template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and platforms commercialized byVisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/IonTorrent, and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S. Pat. Nos.6,210,891; and 6,258,568; each herein incorporated by reference in itsentirety), template DNA is fragmented, end-repaired, ligated toadapters, and clonally amplified in-situ by capturing single templatemolecules with beads bearing oligonucleotides complementary to theadapters. Each bead bearing a single template type is compartmentalizedinto a water-in-oil microvesicle, and the template is clonally amplifiedusing a technique referred to as emulsion PCR. The emulsion is disruptedafter amplification and beads are deposited into individual wells of apicotitre plate functioning as a flow cell during the sequencingreactions. Ordered, iterative introduction of each of the four dNTPreagents occurs in the flow cell in the presence of sequencing enzymesand luminescent reporter such as luciferase. In the event that anappropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10.sup.6 sequencereads can be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55.641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S.Pat. Nos. 6,833,246; 7,115,400; and 6,969,488; each herein incorporatedby reference in its entirety), sequencing data are produced in the formof shorter-length reads. In this method, single-stranded fragmented DNAis end-repaired to generate 5′-phosphorylated blunt ends, followed byKlenow-mediated addition of a single A base to the 3′ end of thefragments. A-addition facilitates addition of T-overhang adapteroligonucleotides, which are subsequently used to capture thetemplate-adapter molecules on the surface of a flow cell that is studdedwith oligonucleotide anchors. The anchor is used as a PCR primer, butbecause of the length of the template and its proximity to other nearbyanchor oligonucleotides, extension by PCR results in the “arching over”of the molecule to hybridize with an adjacent anchor oligonucleotide toform a bridge structure on the surface of the flow cell. These loops ofDNA are denatured and cleaved. Forward strands are then sequenced withreversible dye terminators. The sequence of incorporated nucleotides isdetermined by detection of post-incorporation fluorescence, with eachfluor and block removed prior to the next cycle of dNTP addition.Sequence read length ranges from 36 nucleotides to over 50 nucleotides,with overall output exceeding 1 billion nucleotide pairs per analyticalrun.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbial., 7:287-296; U.S. Pat. Nos. 5,912,148; and 6,130,073; eachherein incorporated by reference in their entirety) also involvesfragmentation of the template, ligation to oligonucleotide adapters,attachment to beads, and clonal amplification by emulsion PCR. Followingthis, beads bearing template are immobilized on a derivatized surface ofa glass flow-cell, and a primer complementary to the adapteroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color, and thus identity of each probe, corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In some embodiments, nanopore sequencing is employed (See, e.g., Astieret al., J. Am. Chem. Soc. 2006 Feb. 8; 128(5)1705-10, incorporated byreference). The theory behind nanopore sequencing relates to what occurswhen a nanopore is immersed in a conducting fluid and a potential(voltage) is applied across it. Under these conditions a slight electriccurrent due to conduction of ions through the nanopore can be observed,and the amount of current is exceedingly sensitive to the size of thenanopore. As each base of a nucleic acid passes through the nanopore,this causes a change in the magnitude of the current through thenanopore that is distinct for each of the four bases, thereby allowingthe sequence of the DNA molecule to be determined.

In some embodiments, HeliScope by Helicos BioSciences is employed(Voelkerding et al., Clinical Chem., 55. 641-658, 2009; MacLean et al.,Nature Rev. Microbial, 7:287-296; U.S. Pat. Nos. 7,169,560; 7,282,337;7,482,120; 7,501,245; 6,818,395; 6,911,345; and 7,501,245; each hereinincorporated by reference in their entirety). Template DNA is fragmentedand polyadenylated at the 3′ end, with the final adenosine bearing afluorescent label. Denatured polyadenylated template fragments areligated to poly(dT) oligonucleotides on the surface of a flow cell.Initial physical locations of captured template molecules are recordedby a CCD camera, and then label is cleaved and washed away. Sequencingis achieved by addition of polymerase and serial addition offluorescently-labeled dNTP reagents. Incorporation events result influor signal corresponding to the dNTP, and signal is captured by a CCDcamera before each round of dNTP addition. Sequence read length rangesfrom 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (See, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appln. Pub.Nos. 2009/0026082; 2009/0127589; 2010/0301398; 2010/0197507;2010/0188073; and 2010/0137143, incorporated by reference in theirentireties for all purposes). A microwell contains a template DNA strandto be sequenced. Beneath the layer of microwells is a hypersensitiveISFET ion sensor. All layers are contained within a CMOS semiconductorchip, similar to that used in the electronics industry. When a dNTP isincorporated into the growing complementary strand a hydrogen ion isreleased, which triggers the hypersensitive ion sensor. If homopolymerrepeats are present in the template sequence, multiple dNTP moleculeswill be incorporated in a single cycle. This leads to a correspondingnumber of released hydrogens and a proportionally higher electronicsignal. This technology differs from other sequencing technologies inthat no modified nucleotides or optics are used. The per base accuracyof the Ion Torrent sequencer is .about.99.6% for 50 base reads, withabout 100 Mb generated per run. The read-length is 100 base pairs. Theaccuracy for homopolymer repeats of 5 repeats in length is about 98%.The benefits of ion semiconductor sequencing are rapid sequencing speedand low upfront and operating costs.

Another exemplary nucleic acid sequencing approach that may be adaptedfor use with the present invention was developed by Stratos Genomics,Inc. and involves the use of Xpandomers. This sequencing processtypically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 2009/0035777, which isincorporated herein in its entirety.

Other single molecule sequencing methods include real-time sequencing bysynthesis using a VisiGen platform (Voelkerding et al., Clinical Chem.,55: 641-58, 2009; U.S. Pat. No. 7,329,492; and U.S. patent applicationSer. No. 11/671,956; and Ser. No. 11/781,166; each herein incorporatedby reference in their entirety) in which immobilized, primed DNAtemplate is subjected to strand extension using a fluorescently-modifiedpolymerase and fluorescent acceptor molecules, resulting in detectiblefluorescence resonance energy transfer (FRET) upon nucleotide addition.

Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55. 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7:287-296; U.S. Pat. Nos.7,170,050; 7,302,146; 7,313,308; and 7,476,503; all of which are hereinincorporated by reference) utilizes reaction wells 50-100 nm in diameterand encompassing a reaction volume of approximately 20 zeptoliters(10.sup.-21 L). Sequencing reactions are performed using immobilizedtemplate, modified phi29 DNA polymerase, and high local concentrationsof fluorescently labeled dNTPs. High local concentrations and continuousreaction conditions allow incorporation events to be captured in realtime by fluor signal detection using laser excitation, an opticalwaveguide, and a CCD camera.

In some embodiments, the single molecule real time (SMRT) DNA sequencingmethods using zero-mode waveguides (ZMWs) developed by PacificBiosciences, or similar methods, are employed. With this technology, DNAsequencing is performed on SMRT chips, each containing thousands ofzero-mode waveguides (ZMWs). A ZMW is a hole, tens of nanometers indiameter, fabricated in a 100 nm metal film deposited on a silicondioxide substrate. Each ZMW becomes a nanophotonic visualization chamberproviding a detection volume of just 20 zeptoliters (10.sup.-21 L). Atthis volume, the activity of a single molecule can be detected amongst abackground of thousands of labeled nucleotides. The ZMW provides awindow for watching DNA polymerase as it performs sequencing bysynthesis. Within each chamber, a single DNA polymerase molecule isattached to the bottom surface such that it permanently resides withinthe detection volume. Phospholinked nucleotides, each type labeled witha different colored fluorophore, are then introduced into the reactionsolution at high concentrations which promote enzyme speed, accuracy,and processivity. Due to the small size of the ZMW, even at these highconcentrations, the detection volume is occupied by nucleotides only asmall fraction of the time. In addition, visits to the detection volumeare fast, lasting only a few microseconds, due to the very smalldistance that diffusion has to carry the nucleotides. The result is avery low background.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. Nos.7,405,281; 7,315,019; 7,313,308; 7,302,146; and 7,170,050; and U.S. Pat.Pub. Nos. 2008/0212960; 2008/0206764; 2008/0199932; 2008/0199874;2008/0176769; 2008/0176316; 2008/0176241; 2008/0165346; 2008/0160531;2008/0157005; 2008/0153100; 2008/0153095; 2008/0152281; 2008/0152280;2008/0145278; 2008/0128627; 2008/0108082; 2008/0095488; 2008/0080059;2008/0050747; 2008/0032301; 2008/0030628; 2008/0009007; 2007/0238679;2007/0231804; 2007/0206187; 2007/0196846; 2007/0188750; 2007/0161017;2007/0141598; 2007/0134128; 2007/0128133; 2007/0077564; 2007/0072196;and 2007/0036511; and Korlach et al. (2008), “Selective aluminumpassivation for targeted immobilization of single DNA polymerasemolecules in zero-mode waveguide nanostructures,” PNAS 105(4): 1176-81,all of which are herein incorporated by reference in their entireties.

EXAMPLE 1

Cell Partitioning and Barcoding in Reversible Polyacrylamide Matrix.

K562 and HEK 3T3 cells are resuspended in a one to one mix and anchoredwith modified L(ipid) M(odified) O(ligonucleotides). This is adaptedfrom McGinnis et al (2019) with the following modification. The 3′ endof the Anchor oligo is modified with acrydite phosphoramidite.

Clonal cell oligo barcode sequences are built on the LMOs as shown inFIG. 1 through split pool synthesis. 1 million of these mixed cells arecombined in a volume of approximately 100 μL of PBS 1× buffer. This cellsuspension is mixed with an equal volume of 12:1 Polyacrylamide: Bac(N,N′-Bis(acryloyl)cystamine) to form a 2% polyacrylamide gelsuspension. After mixing thoroughly with a pipette, Ammonium persulfateand TEMED are added to produce a final concentration of 0.05% and 0.1%,respectively. The mixture is further pipette mixed and the approximately200 μL of solution is deposited in an Eppendorf tube to allow forpolyacrylamide solidification. The acrydite moiety on the Anchor portionof the LMOs will be incorporated in the polyacrylamide matrix duringsolidification to produce a sphere of oligonucleotides at the cellmembrane/hydrogel matrix interface. Once the polyacrylamide issolidified, a cell lysis reagent, such as 0.1% NP40, is added to lysethe cells. Reverse transcription reagents are added and once thereleased mRNA binds to the PolyT track of the PolyT primer, primertemplate reverse transcription occurs to produce barcoded cDNA. DTT isadded to a final concentration of 100 mM to dissolve the Polyacrylamide:BAC matrix to release the barcoded cDNA. The cDNA is collected,purified, concentrated and converted into an NGS library by standardmethods.

EXAMPLE 2

Cell Partitioning and Barcoding in Reversible Agarose Matrix

K562 and HEK 3T3 cells are resuspended in a one to one mix and anchoredwith modified L(ipid) M(odified) O(ligonucleotides). This is adaptedfrom McGinnis et al (2019) with the following modification. The 3′ endof the Anchor oligo is modified with imidazole triphosphate nucleosides(dImTP).

Clonal cell oligo barcode sequences are built on the LMOs as shown inFIG. 2 through split pool synthesis. 1 million of these mixed cells arecombined in a volume of approximately 100 μL of PBS 1× buffer. This cellsuspension is mixed with an equal volume of 2% molten Ni-NTA or Eu-NTAagarose to form a 1% agarose gel suspension. After mixing thoroughly theapproximately 200 μL of solution is deposited in an Eppendorf tube andthe temperature is dropped to allow for agarose solidification. ThedImTP on the anchor portion of the LMOs will bind to the Ni-NTA—orEu-NTA agarose during solidification to produce a sphere ofoligonucleotides at the cell membrane/hydrogel matrix interface. Oncethe agarose is solidified, a cell lysis reagent, such as 0.1% NP40, isadded to lyse the cells. Reverse transcription reagents are added andonce the released mRNA binds to the PolyT track of the PolyT primer,primer template reverse transcription occurs to produce barcoded cDNA.The agarose is heated to melt the matrix and release the barcoded cDNA.The cDNA is collected, purified, concentrated and converted into an NGSlibrary by standard methods.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of tagging cell-specific barcodes tocell nucleic acids, the method comprising, providing (i) cells orisolated cell nuclei having heterologous barcoded oligonucleotidesattached to cell membranes of the cells or (ii) cell nuclei comprisingheterologous oligonucleotides attached to nuclear membranes of theindividual cell nuclei; mixing the cells or nuclei with a liquid-formhydrogel; cross-linking the hydrogel around the cells or nuclei, whereinthe hydrogel forms a solid gel; releasing the heterologous barcodedoligonucleotides from the cell membranes or nuclear membranes togenerate released heterologous barcoded oligonucleotides; allowing theheterologous barcoded oligonucleotides released from the cell membranesor nuclear membranes to locate at solidified hydrogel surrounding thecells or nuclei; attaching the heterologous barcoded oligonucleotides tocell polynucleotides or copies or cDNAs thereof to form barcoded cellpolynucleotides; dissolving the solidified hydrogel or extracting thebarcoded cell polynucleotides from the solidified hydrogel, therebyreleasing barcoded cell polynucleotides from the hydrogel, therebytagging cell-specific barcodes to cell nucleic acids.
 2. The method ofclaim 1, wherein the allowing comprises binding the heterologousbarcoded oligonucleotides released from the cell membranes or nuclearmembranes to the solidified hydrogel surrounding the cells or nuclei. 3.The method of claim 1, wherein the heterologous barcodedoligonucleotides comprise a lipid moiety and wherein the lipid moietyanchors the heterologous barcoded oligonucleotides in the cell membranesor nuclear membranes.
 4. The method of claim 1, wherein the hydrogel iscovalently linked to a molecule having binding affinity for theheterologous oligonucleotides.
 5. The method of claim 4, wherein themolecule is selected from the group consisting of biotin, streptavidin,an antibody, an aptamer, Ni, Eu, or a polynucleotide comprising asequence of at least 6 contiguous nucleotides that is fullycomplementary to a sequence in the heterologous barcodedoligonucleotides.
 6. The method of claim 5, wherein the nuclei comprisefragmented nuclear DNA, wherein the fragmented DNA comprises commonadapter sequences at ends of the fragments.
 7. The method of claim 6,wherein the method further comprises fragmenting the nuclear DNA andintroducing the common adapter sequence with a transposase.
 8. Themethod of claim 1, wherein the hydrogel is covalently linked to amolecule having binding affinity for the heterologous barcodedoligonucleotides and the released heterologous barcoded oligonucleotidesbind to the molecule at a hydrogel/cell interface or hydrogel/nucleiinterface.
 9. The method of claim 1, wherein the releasing comprisescleaving the heterologous barcoded oligonucleotides from a portion ofthe oligonucleotides to free the heterologous barcoded oligonucleotidesfrom the cell membrane or nuclear membrane.
 10. The method of claim 1,wherein the attaching comprises ligating the heterologous barcodedoligonucleotides to cell polynucleotides or copies or cDNAs thereof toform the barcoded cell polynucleotides.
 11. The method of claim 1,wherein the providing comprises synthesizing the heterologous barcodedoligonucleotides on the cells or nuclei using a split and poolmethodology such that different cells or different nuclei are linked toa plurality of identical heterologous barcoded oligonucleotides andwhere different cells or different nuclei have unique heterologousbarcoded oligonucleotides.
 12. The method of claim 1, wherein theallowing comprises diffusion of the heterologous barcodedoligonucleotides released from the cell membranes or nuclear membranesto the solidified hydrogel surrounding the cells or nuclei such that theheterologous barcoded oligonucleotides are localized at ahydrogel/membrane interface.
 13. The method of claim 1, wherein theextracting the barcoded cell polynucleotides from the solidifiedhydrogel comprises electrophoresing the barcoded cell polynucleotidesfrom the solidified hydrogel.
 14. The method of claim 1, furthercomprising sequencing the barcoded cell polynucleotides released fromthe hydrogel.
 15. The method of claim 1, wherein the cells are mammaliancells.
 16. The method of claim 1, wherein the hydrogel comprisesalginate, agarose, polyacrylamide, chitosan, hyaluronan, dextran,collagen, fibrin, polyethylene glycol (PEG), poly(hydroxyethylmethacrylate) (polyHEMA), polyvinyl alcohol (PVA) or polycaprolactone(PCL).
 17. The method of claim 1, wherein the hydrogel comprisesalginate and the crosslinking comprises contacting the hydrogel withcalcium.
 18. The method of claim 12, wherein the molecule isstreptavidin and the heterologous barcoded oligonucleotidesbiotinylated.
 19. The method of claim 12, wherein the molecule is apolynucleotide comprising a sequence of at least 6 contiguousnucleotides that is fully complementary to a sequence in theheterologous barcoded oligonucleotides such that the polynucleotidelinked to the hydrogel hybridizes to the released heterologous barcodedoligonucleotides.
 20. The method of claim 1, wherein the releasingcomprises lysing the cells or nuclei.